Draq5

Tg CypD+, CypD−, and nonTg mouse brains were fixed by perfusion with 4% paraformaldehyde and coronal sections (30 μm) were cut with a Vibratome (Leica VTS1000, Wetzlar Germany). Sections were collected and immersed in wash buffer (0.1 M sodium phosphate, 0.5 M sodium chloride, 0.1% Triton X-100, pH 7.4) for 1 hour. After preincubation with blocking solution (10% normal goat serum, 0.3% Triton X-100 in PBS , pH 7.4) for one hour at room temperature, sections were co-immunostained with primary antibodies [mouse anti-CypD (Ab110324, 1:500; Abcam) and rabbit anti-SODII (1:5000, ADI-SOD-111, Enzo life Sciences, USA) or rabbit anti-MAP2 (1 : 500, A16657, life technologies, USA) at 4 °C for overnight. Sections were then incubated with Alexa Fluor 594-conjugated goat anti-rabbit IgG and 488 goat anti-mouse IgG secondary antibodies (1 : 1000, Invitrogen) for 1 h at room temperature. Nuclei were stained by DRAQ5 (5 μM, Cell Signaling) for 10 min at room temperature. The staining images were taken under confocal microscopy. Brain sections incubated with non-immune IgG or 2^nd antibody alone were used as negative controls. An investigator who was blinded to experimental groups analyzed all images. For quantification of human CypD staining, brain sections were randomly selected and measured using MetaMorph software (Molecular Devices, CA).

The expression of membrane-targeted tandem dimer Tomato (mT) and membrane-targeted green fluorescent protein (mG) was assessed by fluorescence microscopy imaging on intact mesenteric artery segments. Mesenteric artery segments were stained with DRAQ5 (1 μmol/l, Cell Signaling Technology, Danvers, Massachusetts, USA) for 20 min to label the nuclei and washed in PBS three times (20min each). Mesenteric arteries were mounted in Fluoromount (Sigma-Aldrich; St. Louis, Missouri, USA) and imaged using a Wave FX Spinning Disc Confocal microscope (Quorum Technologies; Guelph, Ontario, Canada).